Lecture 19 Summary

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Serine protease mechanism: The role of the catalytic triad, preferential binding of transition state and contribution of a low-barrier hydrogen bond (LBHB). Inhibitor complex (trypsin-BPTI) structure and direct observation of the tetrahedral intermediate. Zymogen forms of the pancreatic serine proteases. (11: 352-360)

Thrombin - yet another serine protease

In the blood clotting cascade, the active form of the serine protease thrombin is responsible for the conversion of fibrinogen to fibrin. A clot forms when fibrin monomers formed by the action of thrombin assemble into ordered fibrous arrays. Thrombin shows a specificity for cleavage of the peptide bond between Arg and Gly, suggesting a similarity to trypsin. Indeed, the B chain of thrombin shows sequence similarity to trypsin, and the X-ray structure of thrombin reveals all the hallmarks of a serine protease: the catalytic triad, oxyanion hole, and specificity pocket. The latter, like trypsin, has an aspartate residue at its bottom that interacts favorably with the Arg at P₁ of the substrate.

Thrombin arises from the processing of an inactive precursor, or zymogen, called prothrombin. Prothrombin is a 582-residue polypeptide whose first 274 residues constitute the large "pro" region. Processing (proteolytic cleavage) by Factor Xa (stimulated by Factor Va) corresponds to cleavage of the Arg274-Thr275 and Arg323-Ile324 peptide bonds. The pro region is released, while the two fragments of the mature polypeptide remain associated and are covalently joined by a disulfide bond.

The pro region of thrombin contains (near the N-terminus) a number of modified Glu residues that contain an extra carboxyl group. These residues, called γ-carboxyglutamate (three-letter abbreviation Gla), act as effective chelators of Ca²⁺ ions. This property of prothrombin is essential for the proper functioning of the clotting cascade. This is because the binding of calcium ions by prothrombin anchors it to phospholipid membranes derived from platelets following injury. This properly localizes prothrombin in proximity to its activating factors.

Learning objectives

Page updated 07-24-2010

References

1. Blevins RA, Tulinsky A. (1985) The refinement and the structure of the dimer of α-chymotrypsin at 1.67 Å resolution. J Biol Chem 260: 4264-.